The present invention related to two cDNA clones, designated to PepDef (pepper defensin protein gene) and PepThi (pepper thionin-like protein gene) and individual component; thereof including its coding region and its gene product; modification thereto; application of said gene, coding region and modification thereto; DNA construct, vectors and transformed plants each comprising the gene or part thereof.
Plants have developed defense mechanisms to defend themselves against phytopathogens. Plants"" first responses to pathogen infection include fortification of cell walls for physical barriers by deposition of lignin (Dean and Kuc, 1988) and by oxidative cross-linking (Brisson et al., 1994) as well as the hypersensitive reaction (HR). HR causes a rapid cell death of infected tissues to halt further colonization by pathogens (Goodman and Novacky, 1994). The next array of defense strategies includes the production of antimicrobial phytoalexins (van Etten et al., 1989), pathogenesis-related (PR) proteins (Linthorst, 1991; Ponstein et al., 1994), and cysteine (Cys)-rich proteins, such as lipid transfer protein (Garcia-Olmedo et al., 1995) and thionins (Bohlmann, 1994).
Thionins are small, highly basic, Cys-rich proteins that show antimicrobial activity and seem to have a role in plant defense against fungi and bacteria. The overexpression of the THI2.1 thionin in Arabidopsis enhanced resistance to a phytopathogenic fungus (Epple et al., 1997). The overexpression of xcex1-hordothionin in tobacco also enhanced resistance to a phytopathogenic bacterium (Carmona et al., 1993). In addition, during barley and powdery mildew interactions, the accumulation of thionins was higher in the incompatible interaction than in the compatible one (Ebrahim-Nesbat et al., 1993).
The thionins contain a signal sequence, the thionin domain and an acid polypeptide domain as well as the conserved Cys residues (Bohlmann et al., 1994). A new class of Cys-rich antimicrobial protein, xcex3-thionin, has a similar size (5 kD) and the same number of disulfide bridges as thionins. However, since xcex3-thionins do not have significant sequence homologies with thionins, they have been described as plant defensins (Terras et al., 1995). Both defensin and thionin genes in Arabidopsis are inducible via a salicylic acid-independent pathway different from that for PR proteins (Epple et al., 1995; Penninckx et al., 1996).
Fruit ripening represents a genetically synchronized process that involves developmental events unique to plant species. Generally, ripe fruits are susceptible to pathogen attack (Swinburne, 1983; Prusky et al., 1991). Therefore, fruit as one of the reproductive organs of the plants must be protected from pathogens to maintain their integrity and seed maturation. Several antifungal proteins that are responsible for protection against pathogens during fruit ripening have been identified (Fils-Lycaon et al., 1996; Meyer et al., 1996; Salzman et al., 1998). Also, PR proteins are developmentally expressed during the formation of flowers (Lotan et al., 1989; Cote et al., 1991).
Colletotrichum gloeosporioides (Penz.) causes anthracnose diseases in many plant species (Daykin, 1984; Dodds et al., 1991; Prusky et al., 1991). C. gloeosporioides is the most prevalent species among C. acutatum, C. coccodes, C. dematium, C. gloeosporioides, and G. cingulata to cause anthracnose diseases on pepper (Capsicum annuum L.) (Kim et al., 1986; Manandhar et al., 1995). In previous study, we found that the unripe-mature-green fruit of pepper cv. Nokkwang interacted compatibly with C. gloeosporioides, whereas the interaction of the ripe-red fruits with fungus was incompatible (Oh et al., 1998). To investigate the activation of defense-related genes from the incompatible-pepper fruit upon C. gloeosporioides infection, we isolated a defensin gene and a thionin-like gene by using mRNA differential display. The regulation of these Cys-rich protein genes was studied during fruit ripening and in the initial infection process during the compatible and incompatible interactions. We report here what appears to be the first case of a defensin gene and a thionin-like gene induced via different signal transduction pathways in a plant and fungus interaction.
The present invention relates to two cDNA clones, designated to a defensin gene, PepDef, and a thionin-like gene, PepThi, the sequences of which are depicted in SEQ ID No. 3 and No. 1, respectively. The anthracnose fungus, C. gloeosporioides, interacts incompatibly with ripe fruits of pepper (Capsicum annuum). It interacts compatibly with the unripe-mature fruits. We isolated PepDef and PepThi expressed in the incompatible interaction by using mRNA differential display method. Both genes were developmentally regulated during fruit ripening, organ-specifically regulated, and differentially induced during the compatible and incompatible interactions. The expression of PepThi gene was rapidly induced in the incompatible-ripe fruit upon fungal infection. The fungal-inducible PepThi gene is highly inducible only in the unripe fruit by salicylic acid. In both ripe and unripe fruits, it was induced by wounding, but not by jasmonic acid. The expression of PepDef gene is enhanced in the unripe fruit by jasmonic acid, while suppressed in the ripe fruit. These results suggest that both small and cysteine-rich protein genes are induced via different signal transduction pathways during fruit ripening to protect the reproductive organs against biotic and abiotic stresses. The PepDef and PepThi car be cloned into an expression vector to produce a recombinant DNA expression system suitable for insertion into cells to form a transgenic plant transformed with these genes. In addition, the PepDef and PepThi genes of this invention can be also used to produce transgenic plants that exhibit enhanced resistance against phytopathogens, including fungi, bacteria, viruses, nematode, mycoplasmalike organisms, parasitic higher plants, flagellate protozoa, and insects.